MicroRNAs (miRNAs) are an abundant and highly conserved class of endogenous ~22-base RNAs that play crucial roles in cell function and development by base pairing to sites within target mRNAs, triggering either translational repression or mRNA degradation, or both. Our long-term goal is to discover the regulatory roles of several conserved miRNAs in critical and well-defined stages in formation of erythrocytes - uncovering not only their specific mRNA targets at both the BFU-E and CFU-E developmental stages but also the underlying network of miRNAs and their mRNA targets essential for these developmental transitions. Our specific aims are 1) to investigate the functions of miR-144, 451, 221, 222, and 223 in terminal proliferation and differentiation of CFU-E progenitors. We will determine the cellular effects on erythroid differentiation of ectopically overexpressing or knocking down expression these miRNAs in cultured CFU-E cells. Next we will determine the developmentally important mRNA targets downregulated by each of these miRNAs during specific stages of CFU-E proliferation and differentiation using a combination of experimental and computational approaches, and we will determine the roles of selected key miRNA target interactions during erythroid differentiation in culture. In Aim 2 we will use similar techniques to determine the functions of miR- 221, miR-222, miR-223 and other developmentally regulated miRNAs in the proliferation of BFU-E progenitors and the formation of CFU-Es. These studies will provide important information on the gene regulatory circuitry that controls hematopoiesis, and provide insights into pathological states caused by aberrant expression of certain miRNAs.